This invention relates to equipment and methods for detecting the presence of, measuring the amount of, and/or monitoring the level of, one or more selected components in a liquid mixture.
While use may be made of this invention in chemical industry, especially where complex mixtures are encountered (e.g. in food chemistry or biochemical engineering) it is of particular value in biological investigation and control techniques. More particularly, it lends itself to animal or human medicine, and in particular to in vivo measuring or monitoring of components in body fluids.
For convenience, the invention will be described, inter alia, with reference to one particular in vivo measurement, the determination of glucose in a diabetic human subject by the use of equipment which, while usable on a specific or occasional basis also lends itself to temporary or permanent implantation. While the provision of sensors for components in biological fluids is one object of the invention, other and broader objects are not hereby excluded.
In vivo glucose sensors have already been proposed. One proposal is based on direct oxidation of glucose at a catalytic platinum electrode (see Hormone and Metabolic Research, Supplement Series No. 8, pp 10-12 (1979)) but suffers from the drawback of being non-specific and of being easily poisoned by interfering substances. Another proposal, for a procedure more specific to glucose, involves the use of glucose oxidase on an oxygen electrode (Adv. Exp. Med. Biol, 50 pp 189-197 (1974) but is not very responsive to the high glucose concentrations. Other systems using glucose oxidase have been proposed but not fully investigated for in vivo methods, see e.g. J. Solid-Phase Biochem. 4 pp 253-262 (1979)).
A fall in the level of oxygen tension resulting from poor tissue perfusion is a particular problem for detecting glucose in blood taken from subcutaneous tissue of diabetics.
The inventors have carried out in vitro studies of enzyme-catalyzed reactions using a mediator (e.g. phenazine methosulfate or phenazine ethosulfate) in solution to transfer the electrons arising from the enzyme, during its action, directly to the electrode, as described in Biotechnology Letters 3 pp 187-192 (1981).
Generally, it is desirable to find a mediator which meets the particularly stringent demands of quantitative electrochemical assaying. For example, the mediator must rapidly transfer electrons between the enzyme and the electrode at a rate representative of the rate of the enzyme-catalysed reaction rate. The mediator should be sensitive to potential differences of the enzyme; however, the mediator's response should be relatively insensitive to the presence of interfering substances. Ideally, the mediator should be capable of effecting electron transfer for a broad range of enzymes and under a broad range of conditions such as temperature and pH. The mediator should not be toxic to cells or carcinogenic.
It is specifically desirable to find an alternative amperometric detection method, based on glucose oxidase, which is not dependent on oxygen as the mediator of electron transfer. Previously described electron acceptors for glucose oxidase include hexacyanoferrate (III), and a range of organic dyes; the former is not readily entrapped at an electrode; the latter, though widely used in spectrophotometric measurements, have a number of disadvantages for electromechanical use including ready autoxidation, instability in the reduced forms and pH-dependent redox potentials.